In this component of the U54 program, promising drug candidates identified in the zebrafish screens described in Project 1 and advanced through in vivo structure activity relationship and medicinal chemistry studies in Project 2 will be tested in progressive translational animal efficacy studies using well-established mouse and rabbit models of cyanide poisoning. We anticipate that Project 2 will yield 4-5 highly promising optimized compounds each year. These candidates will then be tested in mice for in vivo efficacy and for safety in range finding studies. Efficacy will be tested in a well-validated cyanide gas inhalational model. We expect that of the 4-5 drugs tested each year, 2-4 will show promise in mice in terms of safety and efficacy, which will then progress to extensively monitored rabbit efficacy studies to determine physiologic effects, and rate of recovery in addition to survival outcomes. The drugs will be administered by intramuscular injection in both the mice and rabbits, since the CounterACT program is primarily interested in finding a cyanide antidote that could be given rapidly in the field. In addition, candidate drugs that progress to the rabbit model will also be assessed for efficacy via inhalation, since inhaled drugs are rapidly absorbed and this is an effective method to administer drugs to a large number of people. We anticipate that on average one drug per year will make it through the mouse and rabbit studies and be ready for formal toxicological and pharmacokinetic studies. The studies in this project require close collaboration with Project 2 and the Metabolomics Core, but we will also interact with Project 1 to explore mechanisms of drug toxicity during the development phase and to validate markers for specific populations vulnerable to cyanide toxicity.